Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells

Abstract

Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system. (C) 2014 Elsevier Inc. All rights reserved.

Keywords

Endoplasmic reticulum; Membrane protein topology; Protein orientation; GFP; N-linked glycosylation

Published in

Biochemical and Biophysical Research Communications
2014, volume: 450, number: 4, pages: 1587-1592
Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE

SLU Authors

• Presto, Jenny

  • Karolinska Institute

• Johansson, Jan

  • Department of Animal Biosciences, Swedish University of Agricultural Sciences
    
  • Karolinska Institute
  • Tallinn University

UKÄ Subject classification

Biochemistry
Biochemistry and Molecular Biology
Molecular Biology

Publication identifier

• DOI: https://doi.org/10.1016/j.bbrc.2014.07.046

Permanent link to this page (URI)

https://res.slu.se/id/publ/67188